Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL 60614, USA.
Am J Respir Cell Mol Biol. 2010 Sep;43(3):334-41. doi: 10.1165/rcmb.2009-0149OC. Epub 2009 Oct 23.
The cystic fibrosis transmembrane conductance regulator (CFTR) gene is driven by a promoter that cannot alone account for the temporal and tissue-specific regulation of the gene. This has led to the search for additional regulatory elements that cooperate with the basal promoter to achieve coordinated expression. We previously identified two alternative upstream exons of the gene that were mutually exclusive of the first exon, and one of which showed temporal regulation in the human and sheep lung. We now demonstrate that this alternative splice product generates a stable protein, which initiates translation at an ATG in exon 4, and thus lacks the N terminus of CFTR. The other splice variant inhibits translation of the protein. In a search for the promoter used by the upstream exons, we identified a novel element that contributes to the activity of the basal CFTR promoter in airway epithelial cells, but does not function independently. Finally, we demonstrate that, in primary airway cells, skin fibroblasts, and both airway and intestinal cell lines, the CFTR promoter is unmethylated, irrespective of CFTR expression status. Thus, methylation is not the main cause of inactivation of CFTR transcription.
囊性纤维化跨膜电导调节因子 (CFTR) 基因由一个启动子驱动，该启动子本身不能单独解释基因的时间和组织特异性调节。这导致了对额外的调节元件的搜索，这些元件与基础启动子合作以实现协调表达。我们之前鉴定了该基因的两个互斥的上游外显子，其中一个在外显子 4 中表现出时间调节，并且在人和绵羊肺中均表现出时间调节。我们现在证明该选择性剪接产物产生一种稳定的蛋白质，其在 4 号外显子的 ATG 处起始翻译，因此缺乏 CFTR 的 N 端。另一种剪接变体抑制蛋白质的翻译。在寻找上游外显子使用的启动子时，我们鉴定了一个新的元件，该元件有助于气道上皮细胞中基础 CFTR 启动子的活性，但不能独立发挥作用。最后，我们证明在原代气道细胞、皮肤成纤维细胞以及气道和肠细胞系中，CFTR 启动子均未甲基化，而不论 CFTR 表达状态如何。因此，甲基化不是 CFTR 转录失活的主要原因。
